With numerous Gram-negative bacterial species demonstrating antimicrobial drug resistance, the identification of new targets for inhibitor design in bacterial systems is of great importance. Escherichia coli, common Gram-negative bacteria, cause illness in a quarter of a million people and hundreds of deaths each year, in the US alone. As part of our goal of helping to develop new antimicrobial compounds, we have been investigating the heptosyltransferases involved in the biosynthesis of the core region of the lipopolysaccharide (LPS) from Escherichia coli. Heptosyltransferase (Hep) enzymes are essential for the formation of bacterial biofilms in Gram-negative bacteria, making the Hep enzymes an important targets for the development of biofilm inhibitors. Our lab recently demonstrated that E. coli HepI is the first LPS biosynthetic enzyme capable of utilizing fully delipidated substrate analogues while maintaining enzymatic proficiency. Additionally, our work has revealed time-resolved protein dynamics for HepI. This data, along with HepI structural information make this a promising system for the development of inhibitors. Our investigation will address two hypotheses: (1) inhibitor design for this and other glycosyltransferases (GTs) can be improved through characterization of the HepI transition state by kinetic isotope effect studies, and (2) that HepI undergoes significant conformational changes upon ligand binding, the disruption of which might be useful for development of inhibitors for HepI and also other GTs. To date, many inhibitors have been developed for glycosyltransferase enzymes; however, these inhibitors are typically not sufficiently tight-binding for drug development. This proposal seeks to determine whether HepI catalyzes a SN1- or SN2-like reaction in order to enhance our ability to design potent inhibitors for this important class of enzymes. Additionally, since in som enzymological systems including HepI, protein dynamics are necessary for chemistry, efforts to identify inhibitors that not only compete with the substrates, but also those that can disrupt protein dynamics are being pursued. We have recently reported the first transient kinetic analyses on a GT-B protein using stopped-flow fluorescence analyses, and we plan to continue these investigations to allow for determination of whether our inhibitors disrupt chemistry or protein structural changes, like those observed in crystal structures of GTs of the GT-B class. This work promises to enhance drug discovery efforts for multiple systems, including the inhibition of bacterial biofilm formation through inhibition of HepI. 1